Dysfunctional innate immune responsiveness to Porphyromonas gingivalis lipopolysaccharide in diabetes.

INTRODUCTION: Type 1 diabetes is a major risk factor for the development of severe periodontal disease. As diabetes increases in severity, so does the susceptibility to and severity of periodontitis. People with diabetes who have periodontal disease have a harder time maintaining healthy blood glucose levels. Macrophages play an important role in both diabetes and periodontitis. Previous research comparing bone-marrow-derived macrophages (BM-Mvarphi) from diabetic non-obese diabetic (NOD) mice and control mice illustrates that a dysregulation in cytokine, Toll-like receptor (TLR) expression, and cell signaling occurs in the diabetic state. METHODS: This study examines the effect of chronic hyperglycemia on BM-Mvarphi TLR expression and activation, cell signaling, cytokine production, and phagocytic function in the diabetic state, when challenged with the periodontal stimulus Porphyromonas gingivalis lipopolysaccharide (LPS) to further understand how diabetes and associated hyperglycemia may contribute to the increased susceptibility of people with diabetes to periodontitis. RESULTS: When BM-Mvarphi, obtained from diabetic NOD mice, are stimulated with P. gingivalis LPS under hyperglycemic conditions the following changes occur: reduced messenger RNA expression and cell surface expression of TLR2, reduced messenger RNA expression and protein production of tumor necrosis factor-alpha, reduced signal transduction, and a reduction in phagocytic function. All the activity of BM-Mvarphi from diabetic NOD mice was restored when differentiation and stimulation occurred under normoglycemic conditions. DISCUSSION: Diabetic patients in a hyperglycemic state may be generating macrophages that are inherently immunocompromised, contributing to an environment allowing periodontal infections to flourish. As a consequence, people with diabetes who maintain proper control of blood sugar levels may experience an increased immunological benefit when challenged with a periodontal infection.

Fine localization of the CMT4A locus using a PAC contig and haplotype analysis.

Charcot-Marie-Tooth disease type 4A (CMT4A) is a severe, autosomal recessive peripheral neuropathy linked to chromosome 8q13-q21. We have previously constructed a YAC contig across the CMT4A region and narrowed the disease-flanking interval to approximately three megabases. Subsequently, we constructed a PAC/BAC contig made of 44 clones and mapped 44 new and 30 previous STSs, ESTs, and polymorphic makers to the region. Using 13 polymorphic markers, we have now identified an ancestral haplotype segregating in three families, indicating a common founder mutation. Two ancestral recombination events in this haplotype significantly reduce the minimal candidate region to a minimal trailing path of five PAC/BAC clones, which will now allow direct investigation of candidate genes for CMT4A.

Linkage of a gene causing familial focal segmental glomerulosclerosis to chromosome 11 and further evidence of genetic heterogeneity.

Focal segmental glomerulosclerosis (FSGS) is a pathological entity characterized by proteinuria, nephrotic syndrome, and the progressive loss of renal function. It is a common cause of end-stage renal disease (ESRD). Recently, familial forms of FSGS have been identified. Two families with autosomal dominant FSGS were evaluated for linkage using 351 genomic microsatellite markers. Linkage, multipoint analysis, and tests for heterogeneity were performed on the subsequent results. In addition, three small families were used for haplotype analysis. Evidence for linkage was found on chromosome 11q21-q22 for the largest family, with a maximum lod score of 9.89. The gene is currently localized to an 18-cM area between flanking markers D11S2002 and D11S1986. The disease in a second family was not linked to this locus or to a previously described locus on chromosome 19q13. There were no shared haplotypes among affected individuals in the three smaller families. Our findings demonstrate that genetic heterogeneity is prevalent in FSGS in that at least three genes cause the FSGS phenotype. Identification of the genes that cause familial FSGS will provide valuable insights into the molecular basis and pathophysiology of FSGS.

Quantitative DNA pooling to increase the efficiency of linkage analysis in autosomal dominant disease.

DNA pooling is an efficient method to rapidly perform genome-wide linkage scans in autosomal recessive diseases in inbred populations where affected individuals are likely to be homozygous for alleles near the disease gene locus. We wanted to examine whether this approach would detect linkage in autosomal dominant (AD) disorders where affected individuals may share one allele identical by descent at loci tightly linked to the disease. Two large outbred pedigrees in which the AD diseases familial venous malformation (FVM) and hereditary hemorrhagic telangiectasia (HHT1), linked to 9p and 9q, respectively, were investigated. Separate pools of DNA from affected (n = 21 for FVM and 17 for HHT1) and unaffected family members (n = 9 FVM and HHT1), and 25 unrelated population controls were established. Polymorphic markers spanning chromosome 9 at approximately 13.5-cM intervals were amplified using standard PCR. Allele quantitation was performed with a fluorimager. Visual inspection of allele intensities and frequency distributions suggested a shift in frequency of the most common allele in the affecteds lane when compared to control lanes for markers within 30 cM of the FVM and HHT1 loci. These subjective assessments were confirmed statistically by testing for the difference between two proportions (one-sided; P < or = 0.05). When using population controls, the true-positive rates for FVM and HHT1 were 5/5 and 2/5 markers, respectively. False-positive rates for FVM and HHT1 were 3/9 and 2/9, respectively. In both AD diseases investigated, quantitative DNA pooling detected shifts in allele frequency, thus identifying areas of known linkage in most cases. The utility of this technique depends on the size of the pedigree, frequency of the disease-associated allele in the population, and the choice of appropriate controls. Although the false-positive rate appears to be high, this approach still serves to reduce the amount of overall genotyping by about 60%. DNA pooling merits further investigation as a potential strategy in increasing the efficiency of genomic linkage scans.

Analysis of association at single nucleotide polymorphisms in the APOE region.

The discussion of the prospects of using a dense map of single nucleotide polymorphisms (SNPs) to identify disease genes with association analysis has been extensive. However, there is little empiric evidence to support this strategy. To begin to examine the practical issues surrounding this methodology, we identified 10 SNPs in the region immediately surrounding the apolipoprotein E locus (APOE), an established susceptibility gene for Alzheimer disease. Our goal was to examine patterns of allelic association to begin to investigate the question of whether APOE could have been identified using SNPs. Our strongest evidence of association was at the 2 SNPs immediately flanking APOE.

SNPing away at complex diseases: analysis of single-nucleotide polymorphisms around APOE in Alzheimer disease.

There has been great interest in the prospects of using single-nucleotide polymorphisms (SNPs) in the search for complex disease genes, and several initiatives devoted to the identification and mapping of SNPs throughout the human genome are currently underway. However, actual data investigating the use of SNPs for identification of complex disease genes are scarce. To begin to look at issues surrounding the use of SNPs in complex disease studies, we have initiated a collaborative SNP mapping study around APOE, the well-established susceptibility gene for late-onset Alzheimer disease (AD). Sixty SNPs in a 1.5-Mb region surrounding APOE were genotyped in samples of unrelated cases of AD, in controls, and in families with AD. Standard tests were conducted to look for association of SNP alleles with AD, in cases and controls. We also used family-based association analyses, including recently developed methods to look for haplotype association. Evidence of association (P

Confirmation of a second locus for CMT2 and evidence for additional genetic heterogeneity.

The Charcot-Marie-Tooth (CMT) neuropathies are a group of disorders exhibiting neurophysical, pathological and genetic heterogeneity. CMT2 is a diagnostic subtype of this group of disorders characterized by variable expression and age-of-onset and normal or slightly diminished nerve conduction velocities. Previously, linkage and heterogeneity had been reported in CMT2 with linked families localizing to chromosome 1p (CMT2A). Recently a second CMT2 locus has been described on chromosome 7 in a single large CMT2 family (CMT2D). We have performed pedigree linkage analysis on 15 CMT2 families (N = 371 individuals, 106 affected family members) and have confirmed linkage to chromosome 7. Furthermore, using both admixture and multipoint linkage analysis we show conclusive evidence for additional heterogeneity within this clinical subtype with evidence of families that exclude linkage to both the CMT2D and CMT2A regions. In addition, unlike the previous report we found no obvious consistent clinical differences between the linked family types.

Complete genomic screen in Parkinson disease: evidence for multiple genes.

CONTEXT: The relative contribution of genes vs environment in idiopathic Parkinson disease (PD) is controversial. Although genetic studies have identified 2 genes in which mutations cause rare single-gene variants of PD and observational studies have suggested a genetic component, twin studies have suggested that little genetic contribution exists in the common forms of PD. OBJECTIVE: To identify genetic risk factors for idiopathic PD. DESIGN, SETTING, AND PARTICIPANTS: Genetic linkage study conducted 1995-2000 in which a complete genomic screen (n = 344 markers) was performed in 174 families with multiple individuals diagnosed as having idiopathic PD, identified through probands in 13 clinic populations in the continental United States and Australia. A total of 870 family members were studied: 378 diagnosed as having PD, 379 unaffected by PD, and 113 with unclear status. MAIN OUTCOME MEASURES: Logarithm of odds (lod) scores generated from parametric and nonparametric genetic linkage analysis. RESULTS: Two-point parametric maximum parametric lod score (MLOD) and multipoint nonparametric lod score (LOD) linkage analysis detected significant evidence for linkage to 5 distinct chromosomal regions: chromosome 6 in the parkin gene (MLOD = 5.07; LOD = 5.47) in families with at least 1 individual with PD onset at younger than 40 years, chromosomes 17q (MLOD = 2.28; LOD = 2.62), 8p (MLOD = 2.01; LOD = 2.22), and 5q (MLOD = 2.39; LOD = 1.50) overall and in families with late-onset PD, and chromosome 9q (MLOD = 1.52; LOD = 2.59) in families with both levodopa-responsive and levodopa-nonresponsive patients. CONCLUSIONS: Our data suggest that the parkin gene is important in early-onset PD and that multiple genetic factors may be important in the development of idiopathic late-onset PD.